Importance of the Carbohydrate-Binding Module of Clostridium stercorarium Xyn10B to Xylan Hydrolysis

Abstract

The Clostridium stercorarium xylanase Xyn10B is a modular enzyme comprising two thermostabilizing domains, a family 10 catalytic domain of glycosyl hydrolases, a family 9 carbohydrate-binding module (CBM), and two S-layer homologous (SLH) domains [Biosci. Biotechnol. Biochem., 63, 1596-1604 (1999)]. To investigate the role of this CBM, we constructed two derivatives of Xyn10B and compared their hydrolytic activity toward xylan and some preparations of plant cell walls; Xyn10BdeltaCBM consists of a catalytic domain only, and Xyn10B-CBM comprises a catalytic domain and a CBM. Xyn10B-CBM bound to various insoluble polysaccharides including Avicel, acid-swollen cellulose, ball-milled chitin, Sephadex G-25, and amylose-resin. A cellulose binding assay in the presence of soluble saccharides suggested that the CBM of Xyn10B had an affinity for even monosaccharides such as glucose, galactose, xylose, mannose and ribose. Removal of the CBM from the enzyme negated its cellulose- and xylan-binding abilities and severely reduced its enzyme activity toward insoluble xylan and plant cell walls but not soluble xylan. These findings clearly indicated that the CBM of Xyn10B is important in the hydrolysis of insoluble xylan. This is the first report of a family 9 CBM with an affinity for insoluble xylan in addition to crystalline cellulose and the ability to increase hydrolytic activity toward insoluble xylan.

Function of the Family-9 and Family-22 Carbohydrate-Binding Modules in a Modular β-1,3-1,4-Glucanase/Xylanase Derived fromClostridium stercorariumXyn10B

“…Under the assay condition used, family-9 CBM did not promote the hydrolysis of insoluble oat-spelt xylan. This finding is not inconsistent with our previous observation that this CBM increased hydrolytic activity toward low but not high concentrations of insoluble xylan, 7) since the concentration of the substrate used in this study was high enough to be saturated.…”

“…5) A comparison of the enzyme activities of truncated enzymes showed that the family-9 CBM contributed to enhancement of the catalytic activity of the catalytic module toward the low concentration of insoluble xylan. 7) Family-22 CBMs, which are conserved in C. stercorarium Xyn10B, were originally identified and specified as thermostabilizing modules, since their removal from thermophilic enzymes resulted in a decrease in the thermostability and/or optimal temperature of the enzymes. [8][9][10] Later, however, these modules were found to have an affinity for soluble [11][12][13][14] and/or insoluble 15,16) polysaccharides, and were consequently compiled and classified in family-22 of CBMs.…”

Carbohydrate-binding modules: fine-tuning polysaccharide recognition

“…increasing the concentration of the enzyme on the surface of the substrate) leads to more rapid degradation of the polysaccharide [22]. There are numerous examples in the literature where proteolytic excision or genetic truncation of CBMs from the catalytic modules results in significant decreases in the activity of the enzymes on insoluble, but not soluble polysaccharides (see [2,[22][23][24][25][26][27][28][29] for examples). It should be pointed out that there are examples of CBMs that have become components of the substrate-binding sites of glycoside hydrolases, and are pivotal to the substrate specificity and mode of action of the cognate enzymes.…”

Functions of Family-22 Carbohydrate-Binding Modules inClostridium josuiXyn10A

“…Furthermore, the xylanase activity of rCBM-CM was not improved by the presence of family-22 CBMs, although many CBMs have been reported to accelerate the hydrolytic activities of the CMs adjacent to them. [3][4][5][6][7]11,15,16) In conclusion, C. josui Xyn10A is quite different from C. stercorarium Xyn10B in the function of their family-22 CBMs although they are similar in their primary structure, i.e., the CBMs of the former neither contribute to thermostabilization of the CM nor enhance the catalytic activity of the CM toward barley -glucan (-1,3-1,4-glucan). These results clearly indicate that highly homologous enzymes with an identical module organization do not necessarily have similar catalytic properties.…”

“…1) Most of them contain a carbohydrate-binding module (CBM), also grouped into families of CBMs, 2) in addition to a CM. The removal of CBMs from modular glycoside hydrolases such as cellulase and xylanase [3][4][5] or the artificial connection of a CBM and a CM 6,7) often affects enzyme activity towards insoluble and even soluble substrates in a given case. Therefore, the primary role of a CBM is believed to be to place the enzyme on a substrate, resulting in an increase in substrate concentration around the enzyme, and/or to disrupt the crystalline structure of the substrates, thus supplying the CM with more easily digestible substrates.…”